Issues concerning the invention will now be discussed with reference to applications of microRNA (miRNA) expression assays, however, the invention may employ expression assays concerning other non-coding RNA molecules.
miRNAs are single-stranded RNA molecules having a length of around 21 to 23 nucleotides. miRNAs were first described by Victor Ambros in 1993 and since then over 2,000 papers on have been published on the subject of miRNAs. There are predicted to be about 1,000 miRNAs in humans of which around 600 have been described and experimentally validated to date, although some estimates place the figure at tens of thousands. However, a recent report, which sought to produce an expression atlas of miRNA in various human and rodent tissues and cell lines, reported that around 300 miRNAs accounted for 97% of all detected miRNAs.
miRNA is not translated into protein but instead regulates the expression of one or more other genes. Known biology currently shows that microRNAs target particular individual messenger RNAs (mRNAs) or groups of mRNAs, thereby preventing their translation or, less frequently, accelerating mRNA degradation. The mature single stranded miRNA molecule complexes with the RNA-Induced Silencing Complex (RISC) protein and binds to a partially complementary sequence within the 3′ untranslated region (3′-UTR) of the protein coding mRNA from its target gene. Further proteins are recruited to form a silencing complex and the expression of the target gene product is repressed by a mechanism that blocks the translation of the mRNA.
Although much remains to be discovered about the biology of miRNAs and the composition and mechanism of the silencing complex it is apparent that miRNAs are involved in the regulation of many genes. MiRNAs are thought to regulate as many as 30% of all genes (Xie et al, 2005) at the translational level. An miRNA can regulate multiple genes and each gene can be regulated by multiple miRNAs. Tissue-specific expression of miRNAs is thought to guide commitment of cells to differentiate and/or actively maintain tissue identity. This wide-ranging influence and interplay between different miRNAs suggests that deregulated expression of a single miRNA or small sub-set of miRNAs may result in complex disease traits (Lim et al, 2005, Nature). More than 50% of known human miRNAs reside in genomic regions prone to alteration in cancer cells (Calin et al, 2004 PNAS, 101, 299-3004). Not surprisingly, the expression pattern of miRNAs change in cancer and other disease states. This information has begun to be used to classify and stage cancers, reveal biomarkers for prognosis and response and provide a critical determinant to guide therapeutic intervention, explain chemosensitivity and inform the mechanisms of chemoresistance by allowing the definition of specific miRNA expression patterns in cancer stem cells.
Applications of miRNAs to research and the development of possible new therapeutics have typically resulted from detailed and time consuming analysis of the mechanisms by which miRNA expression and processing is regulated and the mechanisms by which specific miRNAs regulate mRNA translation. Specific drug targets have been identified and research in connection with these drug targets in ongoing. However, although thorough, this research paradigm is time consuming and expensive.
Thus, the invention aims to provide alternative methods for discovering practical applications of interventions, such as the administration of a therapeutic agent, which do not require a detailed understanding of the mechanism of action of the intervention or the identification of a specific drug target. Some embodiments of the invention address the problem of determining new indications for known therapeutic entities or predicting pharmacological properties of test agents, such as aspects of their toxicological profile.